Breast cancer continues to have a devastating effect in terms of mortality and quality of life, especially in the case of recurrent or metastatic disease. Local or regional recurrences and metastatic disease have unfavorable prognosis, and distant metastases are mostly treated with palliative rather than curative approaches. It is only too apparent that, although our current paradigms and treatments for cancer have resulted in substantial progress, the disease frequently evades control and cure. It is therefore imperative to search for new possibilities in the etiology, progression and treatment of cancer, and to understand causes for recurrence and metastasis. The discovery of subpopulations of breast cancer cells with stem-like characteristics is offering new paradigms for understanding and treating tumor recurrence and metastasis. The unique physiological and metabolic environments of solid tumors such as hypoxia, acidic extracellular pH, and altered choline metabolism can influence tumor progression, metastasis and response to therapy. Recent studies suggest that hypoxia provides a niche for stem cells to maintain their precursor status. Hypoxia is also a major cause of radiation and chemo-resistance. We intend to understand the role of hypoxia and choline metabolism in harboring or creating stem-like breast cancer cells. We will target hypoxia and choline metabolism and determine if these interventions reduce the stem-like cell burden in tumors, and reduce metastasis. The aims and hypotheses will be tested using invasive estrogen receptor/progesterone receptor (ER/PR) negative MDA-MB-231 human and noninvasive ER/PR positive MCF-7 human breast cancer cell lines stably expressing enhanced green or red fluorescence protein under control of a hypoxia response element (HRE). Co-registered maps of vascular volume, permeability, and total choline will be obtained with MR. Hypoxic regions will be identified by fluorescence microscopy. Stem-like cancer cells will be identified in vivo by SPECT imaging of CD44/CD24 expression and by immunostaining of fresh/frozen tissue slices. These studies will (i) provide insight into interactions between the tumor microenvironment and stem-like breast cancer cells, (ii) lead to image-guided strategies targeting specific microenvironmental or metabolic niches harboring stem-like breast cancer cells, and (iii) identify non-invasive clinically translatable imaging parameters to detect regions harboring stem-like breast cancer cells. PUBLIC HEALTH RELEVANCE: discovery of stem-like cancer cells in breast cancer is offering new paradigms for understanding and treating recurrence and metastasis. Noninvasive imaging is particularly important in tracking the dynamics of this subpopulation especially for therapies targeting stem-like cancer cells. Our focus is to understand the role of hypoxia, choline metabolism, and the tumor microenvironment in creating or harboring stem-like breast cancer cells, and the effect of targeting hypoxia and choline metabolism on this subpopulation. These studies will identify noninvasive surrogate markers for tumor regions containing stem-like breast cancer cells, and create novel treatments to target this lethal subpopulation.